Methods of treating severe acute respiratory syndrome

ABSTRACT

The disclosure is directed to methods of using dantrolene or a dantrolene prodrug, or a pharmaceutically acceptable salt thereof, to treat Severe Acute Respiratory Syndrome.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 63/008,529, filed Apr. 10, 2020 and U.S.Provisional Patent Application No. 63/062,623, filed Aug. 7, 2020, thedisclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The disclosure is directed to methods of using dantrolene or adantrolene prodrug, or a pharmaceutically acceptable salt thereof, totreat severe acute respiratory syndrome.

BACKGROUND

Severe Acute Respiratory Syndrome (SARS) is a viral respiratory illnesscaused by a SARS-associated coronavirus (SARS-CoV), for example,SARS-CoV-1 and SARS-CoV-2. SARS-CoVs pose a threat to worldwide publichealth. Development of treatments effective to treat these viruses isneeded.

SUMMARY

The disclosure is directed to methods of treating Severe AcuteRespiratory Syndrome in a subject comprising administering to thesubject dantrolene or a pharmaceutically acceptable salt thereof or byadministering a dantrolene prodrug or a pharmaceutically acceptable saltthereof.

The disclosure is also directed to methods for inhibiting replication ofSARS-CoV in a subject comprising administering to the subjectdantrolene, or a pharmaceutically acceptable salt thereof or byadministering a dantrolene prodrug or a pharmaceutically acceptable saltthereof.

The disclosure is also directed to methods for inhibiting replication ofSARS-CoV in a host cell, inhibiting entry of SARS-CoV into a host cell,inhibiting SARS-CoV virion maturation in a host cell, or inhibitingrelease of SARS-CoV from a host cell comprising administering to thehost cell dantrolene, or a pharmaceutically acceptable salt thereof orby administering a dantrolene prodrug or a pharmaceutically acceptablesalt thereof.

The disclosure is also directed to methods for reducing the infectivityof SARS-CoV by administering to a host cell dantrolene, or apharmaceutically acceptable salt thereof or by administering adantrolene prodrug or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure may be understood more readily by reference tothe following detailed description taken in connection with theaccompanying figures and examples, which form a part of this disclosure.It is to be understood that this disclosure is not limited to thespecific compositions, devices, methods, applications, conditions, orparameters described and/or shown herein, and that the terminology usedherein is for the purpose of describing particular embodiments by way ofexample only and is not intended to be limiting of the claimeddisclosure.

As used in the specification including the appended claims, the singularforms “a,” “an,” and “the” include the plural, and reference to aparticular numerical value includes at least that particular value,unless the context clearly dictates otherwise.

When a range of values is expressed, an exemplary embodiment includesfrom the one particular value and/or to the other particular value. Allranges are inclusive and combinable. Further, reference to values statedin ranges includes each and every value within that range. When valuesare expressed as approximations, by use of the preposition “about,” itwill be understood that the particular value forms another embodiment.The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass reasonable variations of the value, such as, for example, ±10%from the specified value. For example, the phrase “about 50%” caninclude ±10% of 50, or from 45% to 55%, inclusive of 50%.

It is to be appreciated that certain features of the disclosure whichare, for clarity, described herein in the context of separateembodiments, may also be provided in combination in a single embodiment.Conversely, various features of the disclosure that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination.

As used herein, whether by itself or in conjunction with another term orterms, it should be understood that the phrases “method of treating” and“method of treatment” may be used interchangeably with the phrase “foruse in the treatment of” a particular disease.

As used herein, whether by itself or in conjunction with another term orterms, “pharmaceutically acceptable” indicates that the designatedentity such as, for example, a pharmaceutically acceptable excipient, isgenerally chemically and/or physically compatible with other ingredientsin a composition, and/or is generally physiologically compatible withthe recipient thereof.

As used herein, whether by themselves or in conjunction with anotherterm or terms, “subject(s),” “individual(s),” and “patient(s)”, refer tomammals, including humans. The term human(s) refers to and includes, ahuman child, adolescent, or adult.

As used herein, whether by themselves or in conjunction with anotherterm or terms, “treats,” “treating,” “treated,” and “treatment,” referto and include ameliorative, palliative, and/or curative uses andresults, or any combination thereof. In other embodiments, the methodsdescribed herein can be used prophylactically. It should be understoodthat “prophylaxis” or a prophylactic use or result do not refer to norrequire absolute or total prevention (i.e., a 100% preventative orprotective use or result). As used herein, prophylaxis or a prophylacticuse or result refers to uses and results in which administration of acompound or composition diminishes or reduces the severity of aparticular condition, symptom, disorder, or disease described herein;diminishes or reduces the likelihood of experiencing a particularcondition, symptom, disorder, or disease described herein; or delays theonset or relapse (reoccurrence) of a particular condition, symptom,disorder, or disease described herein; or any combination of theforegoing.

As used herein, whether used alone or in conjunction with another termor terms, “therapeutic” and “therapeutically effective amount” refer toan amount of a compound or composition that (a) treats a particularcondition, symptom, disorder, or disease described herein; (b)attenuates, ameliorates, or eliminates one or more symptoms of aparticular condition, disorder, or disease described herein; (c) delaysthe onset or relapse (reoccurrence) of a particular condition, symptom,disorder, or disease described herein. It should be understood that theterms “therapeutic” and “therapeutically effective” encompass any one ofthe aforementioned effects (a)-(c), either alone or in combination withany of the others (a)-(c).

As used herein, a “host cell” is, for example, an epithelial cell, forexample, a pulmonary epithelial cell, for example, a mammalian pulmonaryepithelial cell such as a human pulmonary epithelial cell. Other hostcells include white blood cells, for example, macrophages and T-cells.

As used herein, “normalization of fever” is reduction of a subject'stemperature to <36.6° C. armpit, <37.2° C. oral, or <37.8° C. rectal,sustained for at least 24 hours.

As used herein, “normalization of oxygen saturation” is an increase in asubject's peripheral capillary oxygen saturation (SpO2)>94%, sustainedfor at least 24 hours.

As used herein, “inhibiting replication of SARS-CoV” refers todecreasing viral load of SARS-CoV. Methods for determining SARS-CoVreplication inhibition can be determined by those skilled in the art.

Dantrolene is approved for treating malignant hyperthermia andpreventing malignant hyperthermia in high-risk patients. Malignanthyperthermia is a condition that predisposes susceptible individuals toa life-threatening adverse reaction upon exposure to potent volatileanesthetics (halothane, isoflurane, sevoflurane, desflurane, etc.) andthe skeletal muscle relaxant succinylcholine. The anesthetic drugstrigger an uncontrolled Ca²⁺ release from the endoplasmic reticulum (ER)through the ryanodine receptors (RyR) causing a rapid and sustained risein myoplasmic Ca²⁺. Administration of dantrolene reestablishes cellularcalcium homeostasis by inhibiting the release channels in the ER,resulting in lower levels of intracellular Ca²⁺.

RYANODEX (dantrolene sodium, 250 mg/vial) is approved for treatingmalignant hyperthermia and for preventing malignant hyperthermia inhigh-risk patients. RYANODEX forms an aqueous nanosuspension for IVinjection containing dantrolene 50 mg/mL upon reconstitution with 5 mLof USP sterile water for injection (WFI) (without a bacteriostaticagent). Dissolution of RYANODEX suspension in human plasma is extremelyrapid, achieving complete dissolution within 1 minute.

Dantrolene is a surprisingly effective treatment for SAR-CoV infection.Dantrolene can decrease the virus' ability to replicate, mature, createvirions, release from cells, and/or infect other cells.

Methods of the disclosure can also be accomplished using dantroleneprodrugs, and pharmaceutically acceptable salts thereof. Exemplarydantrolene prodrugs are described in WO2019/079721, the entirety ofwhich is incorporated by reference herein.

Preferred dantrolene prodrugs include, for example, compounds of formulaI

wherein R is —P(O)(OH)₂ or —P(O)(OR₁)(OR₂); R₁ is H, —C₁₋₂₆alkyl, aryl,C₁₋₆alkC(O)O—C₁₋₂₆alkyl, —C₁alkOC(O)C₁₋₂₆alkyl, orC₁alkOC(O)OC₁₋₂₆alkyl; and R₂ is —C₁₋₂₆alkyl, aryl,C₁₋₆alkC(O)O—C₁₋₂₆alkyl, —C₁alkOC(O)C₁₋₂₆alkyl, orC₁alkOC(O)OC₁₋₂₆alkyl, as well as pharmaceutically acceptable saltsthereof. Particularly preferred compounds of formula I include compounds2 and 2a:

Other dantrolene prodrugs include compounds of formula II

wherein R₃ is H, —C(O)—Z—N(R₄)(R₅), —C(O)Z—C(O)—OH, or—C(O)—NH—Y—CH₂—OC(O)—Z—C(O)—OH; Z is C₁₋₆alk; Y is arylene; C₁₋₆alkyl;R₅ is H or C₁₋₆alkyl; or R₄ and R₅, together with the nitrogen to whichthey are attached, form a heterocycloalkyl; as well as pharmaceuticallyacceptable salts thereof.

One aspect of the invention is directed to methods of treating SARS in asubject. The subject may be clinically diagnosed with SARS. Criteria fordiagnosing a subject with SARS are known and include laboratoryconfirmation of a SARS-CoV infection as determined using PCR, incombination with presentation of one or more SARS symptoms. Other assaysfor determining SARS-CoV infections can also be used. Symptoms of SARSinclude mild to severe respiratory illness with symptoms of fever,cough, and shortness of breath. Some SARS patients may develop pneumoniain one or both lungs. Some SARS patients may develop multi-organfailure.

In some aspects, the subject may be suspected of having SARS, based on,for example, having experienced close contact with another person whohas been clinically diagnosed with SARS or who has been clinicallydiagnosed with a SARS-CoV infection. Other subjects may be suspected ofhaving SARS based on the subject's symptom presentation.

In some aspects, the subject is treated for SARS by administering to thesubject dantrolene. In other aspects, the subject is treated for SARS byadministering to the subject a pharmaceutically acceptable salt ofdantrolene, for example, dantrolene sodium. In some aspects, the subjectis treated for SARS by administering to the subject a dantroleneprodrug, for example, Compound 2. In some aspects, the subject istreated for SARS by administering to the subject a salt of a dantroleneprodrug, for example, Compound 2a. Administration is preferably of atherapeutically effective amount of the dantrolene, pharmaceuticallyacceptable salt of dantrolene, dantrolene prodrug, or salt of adantrolene prodrug. Therapeutically effective amounts include, forexample, about 1 mg/kg to 10 mg/kg, administering daily, for one or moredays. Particularly preferred amounts include about 1, 1.5, 2, 2.5, 3,3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 mg/kg,administered daily, in one or more doses, for one or more days.

In some aspects, the administration results in at least a 1-pointdecrease in the subject's WHO Ordinal Scale score, as compared to thesubject's WHO Ordinal Scale score at baseline. Methods of assessing WHOOrdinal Scale score are known in the art. In some aspects, theadministration results in a 2-point decrease in the subject's WHOOrdinal Scale score, as compared to the subject's WHO Ordinal Scalescore at baseline. In some aspects, the administration results in a3-point decrease in the subject's WHO Ordinal Scale score, as comparedto the subject's WHO Ordinal Scale score at baseline. In some aspects,the administration results in a 4-point decrease in the subject's WHOOrdinal Scale score, as compared to the subject's WHO Ordinal Scalescore at baseline. In some aspects, the administration results in a5-point decrease in the subject's WHO Ordinal Scale score, as comparedto the subject's WHO Ordinal Scale score at baseline.

In some aspects, the administration results in an improvement, forexample, an increase in the subject's Sequential Organ FailureAssessment daily score, as compared to baseline. Methods of assessing asubject's Sequential Organ Failure Assessment daily score are known inthe art.

In some aspects, the administration results in a reduction of time tonormalization of fever in the subject, as compared to the amount of timeto normalization of fever in a control subject, for example, as comparedto a subject who has only received standard of care treatment. In someaspects, the administration results in a reduction of fever in thesubject treated for SARS. In other aspects, the administration resultsin a clinically significant reduction of fever in the subject. In someaspects, the administration results in a normalization of fever in thesubject.

In some aspects, the administration results in a reduction of time tonormalization of oxygen saturation in the subject, as compared to theamount of time to normalization of oxygen saturation in a controlsubject, for example, as compared to a subject who has only receivedstandard of care treatment. In some aspects, the administration resultsin an increase of oxygen saturation in the subject. In other aspects,the administration results in a clinically significant increase inoxygen saturation in the subject. In other aspects, the administrationresults in normalization of oxygen saturation in the subject.

In yet other aspects, the administration results in improvement in oneor more symptoms of SARS in the subject. In other aspects, theadministration results in a clinically significant improvement in one ormore symptoms of SARS in the subject.

Other aspects of the disclosure are directed to methods of inhibitingreplication of SARS-CoV in a subject. The subject may be clinicallydiagnosed with a SARS-CoV infection. Criteria for diagnosing a subjectwith a SARS-CoV infection are known and include laboratory confirmationas determined using PCR. Other assays for determining SARS-CoVinfections can also be used.

In some aspects, the subject may be suspected of having a SARS-CoVinfection, based on, for example, having experienced close contact withanother person who has been clinically diagnosed with SARS or who hasbeen clinically diagnosed with a SARS-CoV infection.

In some aspects, inhibition of SARS-CoV replication in a subject isaccomplished by administering to the subject dantrolene. In otheraspects, inhibition of SARS-CoV replication in a subject is accomplishedby administering to the subject a pharmaceutically acceptable salt ofdantrolene, for example, dantrolene sodium. In some aspects, inhibitionof SARS-CoV replication in a subject is accomplished by administering tothe subject a dantrolene prodrug, for example, Compound 2. In someaspects, inhibition of SARS-CoV replication in a subject is accomplishedby administering to the subject a salt of a dantrolene prodrug, forexample, Compound 2a. Administration is preferably of a therapeuticallyeffective amount of the dantrolene, pharmaceutically acceptable salt ofdantrolene, dantrolene prodrug, or salt of a dantrolene prodrug.Therapeutically effective amounts include, for example, about 1 mg/kg to10 mg/kg, administering daily, for one or more days. Particularlypreferred amounts include about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or about 10 mg/kg, administered daily,in one or more doses, for one or more days.

Some aspects of the disclosure are directed to methods for inhibitingreplication of SARS-CoV in a host cell. Inhibition of viral replicationcan be determined by those skilled in the art. In these methods,replication is inhibited by administering dantrolene to the host cell.In other aspects, replication is inhibited by administering apharmaceutically acceptable salt of dantrolene to the host cell, forexample, dantrolene sodium. In some aspects, replication is inhibited byadministering to the host cell a dantrolene prodrug, for example,Compound 2. In some aspects, replication is inhibited by administeringto the host cell a salt of a dantrolene prodrug, for example, Compound2a.

Some aspects of the disclosure are directed to methods for inhibitingentry of SARS-CoV into a host cell. Inhibition of viral entry into ahost cell can be determined by those skilled in the art. In thesemethods, viral entry is inhibited by administering dantrolene to thehost cell. In other aspects, viral entry is inhibited by administering apharmaceutically acceptable salt of dantrolene to the host cell, forexample, dantrolene sodium. In some aspects, viral entry is inhibited byadministering to the host cell a dantrolene prodrug, for example,Compound 2. In some aspects, viral entry is inhibited by administeringto the host cell a salt of a dantrolene prodrug, for example, Compound2a.

Some aspects of the disclosure are directed to methods for inhibitingSARS-CoV virion maturation in a host cell. Inhibition of virionmaturation in a host cell can be determined by those skilled in the art.In these methods, virion maturation is inhibited by administeringdantrolene to the host cell. In other aspects, virion maturation isinhibited by administering a pharmaceutically acceptable salt ofdantrolene to the host cell, for example, dantrolene sodium. In someaspects, virion maturation is inhibited by administering to the hostcell a dantrolene prodrug, for example, Compound 2. In some aspects,virion maturation is inhibited by administering to the host cell a saltof a dantrolene prodrug, for example, Compound 2a.

Some aspects of the disclosure are directed to methods for release ofSARS-CoV from a host cell. Inhibition of release from a host cell can bedetermined by those skilled in the art. In these methods, viral releaseis inhibited by administering dantrolene to the host cell. In otheraspects, viral release is inhibited by administering a pharmaceuticallyacceptable salt of dantrolene to the host cell, for example, dantrolenesodium. In some aspects, viral release is inhibited by administering tothe host cell a dantrolene prodrug, for example, Compound 2. In someaspects, viral release is inhibited by administering to the host cell asalt of a dantrolene prodrug, for example, Compound 2a.

Some methods of the disclosure are directed to methods for reducing theinfectivity of SARS-CoV. Reduction of infectivity can be determined bythose skilled in the art. In these methods, infectivity is reduced byadministering dantrolene to the host cell. In other aspects, infectivityis reduced by administering a pharmaceutically acceptable salt ofdantrolene to the host cell, for example, dantrolene sodium. In someaspects, infectivity is reduced by administering to the host cell adantrolene prodrug, for example, Compound 2. In some aspects,infectivity is reduced by administering to the host cell a salt of adantrolene prodrug, for example, Compound 2a.

The following examples are provided to illustrate some of the conceptsdescribed within this disclosure. While each example is considered toprovide specific individual embodiments of disclosure, none of theExamples should be considered to limit the more general embodimentsdescribed herein. In the following examples, efforts have been made toensure accuracy with respect to numbers used (e.g. amounts, temperature,etc.) but some experimental error and deviation should be accounted for.

EXAMPLES Example 1 Methodology

The study is a single-center, open-label, two-arm parallel study ofdantrolene for the adjuvant treatment of COVID-19 administeredintravenously (IV). In one treatment arm, dantrolene will beadministered in conjunction with current standard of care followingmedical practice and procedures established for the in-hospitaltreatment of patients with COVID-19. In the second treatment arm,subjects will receive current standard of care following medicalpractice and procedures established for the in-hospital treatment ofpatients with COVID-19.

Following initial triage and primary assessment of a subject, thesubject's baseline status will be documented. Once eligibility criteriaand baseline status are obtained, administration of dantrolene will beinitiated.

Group A: dantrolene, in addition to standard of care

Group B: standard of care only

Treatment Administration—Group A

Eligible COVID-19 subjects randomized to Group A will receive dantrolene(as RYANODEX, dantrolene sodium) as follows:

-   -   A 1 mg/kg dose will be administered as IV push approximately        every 12 hours for 2 consecutive days (Study Day 1 and Day 2).    -   On Day 3 of the Study, subjects showing adequate tolerability to        1 mg/kg dose will start receiving 2 mg/kg dose as IV push        approximately every 12 hours for the remainder of the study        (Study Day 3 to Day 14, inclusive). Adequate tolerability is        defined as lack of clinically significant adverse reactions that        may have a negative impact on the subject's overall status that        are not secondary to COVID-19, any other underlying condition or        concomitant medication.    -   If a patient does not show adequate tolerability to the 2        mg/dose, the subject will be discontinued from the study.    -   Each subject participating in the study will receive up to 4        (four) 1 mg/kg doses (Days 1-2) and up to 24 (twenty four) doses        of 2 mg/kg (Days 3-14), each administered as an IV push (up to a        minute).

Subjects receiving dantrolene will continue to receive all othertreatments as prescribed, with the exception of treatments included inthe Exclusion Criteria. Assessment of vital signs (blood pressure, heartrate, respiratory rate, body temperature) should be clearly recordedprior to and within 10 minutes after administration of each dose.

Group B

Patients randomized to Group B will receive standard of care, followingacceptable medical practice.

Study Phases

The study will include 2 phases: Screening and Treatment.

During the Screening Phase, eligibility and baseline assessment will beperformed. Informed Consent will be obtained prior to initiation ofstudy procedures.

After determination of eligibility and obtaining Informed Consent,eligible patients will be randomized to Group A or Group B and theTreatment Phase will be initiated and will proceed with administrationof Study Drug as indicated above to Group A Non-eligible subjects willreceive medical assistance as deemed necessary by the attendingphysician following accepted medical practices.

Stopping the Study Drug

If the subject demonstrates clinically significant signs/symptoms ofdantrolene toxicity, the study drug should be stopped. Dantrolenetoxicity may include muscular weakness and alterations in the state ofconsciousness (e.g., lethargy, sedation), vomiting, diarrhea, andcrystalluria, which are not attributable to other cause, such asprogression of COVID-19, other underlying conditions (e.g., sepsis,hypoxia, uncontrolled diabetes) and/or concomitant medications (e.g.,sedatives, antibiotics, antipyretics). The study drug can be stopped atany time.

Diagnosis and Main Criteria for Inclusion

Male or non-pregnant female subjects will be entered into the study ifthey are diagnosed with COVID-19 and meet all the following criteria atScreening:

-   -   At least 18 years of age.    -   Willing and able to provide written informed consent prior to        performing study procedures, or an authorized representative is        willing and able to provide consent on behalf of the patient if        he/she is unable to do so.    -   COVID-19 severity score 3-5 according to the WHO Ordinal Scale        of Severity    -   COVID-19 symptoms onset within 7 days prior to Screening.    -   Hospitalized patient.    -   Has laboratory-confirmed SARS-CoV-2 infection as determined by        PCR, or other commercial or public health assay within 48 hours        prior to Screening.    -   Febrile defined as temperature ≥36.6° C. armpit, ≥37.2° C. oral,        or ≥37.8° C. rectal documented at least within 48 hours of        consent.

Male or non-pregnant female subjects will be excluded from entering thisstudy if they meet any of the following criteria at Screening:

-   -   Participation in any other clinical trial of an experimental        treatment for COVID-19.    -   Alanine Aminotransferase (ALT) or aspartate aminotransferase        (AST) >5× upper limit of normal (ULN).    -   Pregnant women or women who are breastfeeding.    -   Presence of comorbidities that imply a poor prognosis (according        to clinical judgment).    -   Allergy to dantrolene.

Dosage and Mode of Administration

Ryanodex: (dantrolene sodium) for injectable suspension; 250 mg/vial tobe reconstituted with 5 mL of sterile water for injection (without abacteriostatic agent) to yield a 50 mg/mL suspension; to be administeredas a rapid IV push of 1 mg/kg or 2 mg/kg, as described in the protocol.

Efficacy will be evaluated on criteria including:

-   -   World Health Organization (WHO) Ordinal Scale of Severity score.    -   Sequential Organ Failure Assessment (SOFA) score.    -   Length of time to normalization of fever (fever normalization as        defined by temperature <36.6° C. armpit, <37.2° C. oral, or        <37.8° C. rectal sustained for minimum of 24 hours).    -   Length of time to normalization of oxygen saturation (oxygen        normalization as defined by peripheral capillary oxygen        saturation (SpO2) >94% sustained for at least 24 hours). Change        in clinical status at Day 14 using the WHO Ordinal Scale (1-8        score), compared to Baseline.        -   1: Ambulatory. No limitations of activities        -   2: Ambulatory. Limitation of activities        -   3: Hospitalized, Mild Disease. No oxygen therapy        -   4: Hospitalized, Mild Disease. Oxygen by mask or nasal            prongs        -   5: Hospitalized, Severe Disease. Non-invasive ventilation or            high-flow oxygen        -   6: Hospitalized, Severe Disease. Intubation and mechanical            ventilation        -   7: Hospitalized, Severe Disease. Ventilation+additional            organ support (pressors, renal replacement therapy,            extracorporeal membrane oxygenation)        -   8: Death    -   Change in clinical status at Day 5 and Day 10 using the WHO        Ordinal Scale (1-8 score), compared to Baseline.    -   Time to a 1-point decrease in the WHO Ordinal Scale score.    -   Change in Sequential Organ Failure Assessment (SOFA) daily score        (Days 1-14) compared to Baseline.    -   Length of time to normalization of fever (fever normalization as        defined by temperature <36.6° C. armpit, <37.2° C. oral, or        <37.8° C. rectal sustained for minimum of 24 hours).    -   Length of time to normalization of oxygen saturation (oxygen        normalization as defined by peripheral capillary oxygen        saturation (SpO2) >94% sustained for at least 24 hours).

Safety will be evaluated and will include measurement and observation of(if any):

-   -   Vital signs (heart rate, blood pressure, respiratory rate, body        temperature)    -   Clinical laboratory tests (hematology and blood chemistry)    -   ECG monitoring    -   Oxygen saturation    -   Physical exam

Example 2

Dantrolene concentrations were tested: 5, 10, 20, 30, 40, 50 and 100 μM(as RYANODEX, dantrolene sodium).

Testing against a SARS-CoV-2 virus, represents the etiologic agent ofthe COVID-19 global pandemic. Testing was via a standard virusneutralization (VN) assay, which assessed ability to neutralizeSARS-CoV-2.

Virus Neutralization (VN) Assay. The VN assay was performed using VeroE6 cells which are susceptible to SARS-CoV-2 infection. The cells wereseeded into 96-well plates one (1) to three (3) days prior to VN assay,and were incubated at 37° C. and 5% CO₂. On the day of assay, themonolayer of Vero E6 cells was at least 70% to 80% confluent in order torun the VN. The preparation of the compound for VN was performed the dayof assay. The compound was diluted with serum-free media to the desiredstarting concentration (1:10) and then serially diluted 2-fold inserum-free media.

Standardization of the virus required that a TCID50 had previously beenrun to determine the concentration of infectious virus particle per mLof virus stock. Using the TCID50 titer, calculations were performed todefine how much serum-free media to add to the virus stock to yield 1e2TCID50/mL. Once the standardized virus was made, an equal volume of itwas added to the deep 96-well plate containing the diluted compoundsamples. Incubation was maintained for at least one hour. Thevirus/compound mixture was then transferred from the deep 96-well racksinto the appropriate Vero-seeded 96-well plates. After this addition,the plates were returned to the 37° C. and 5% CO₂ incubator for three(3) to five (5) days to six (6) days. After the incubation period, thewells were observed under a phase contrast inverted scope and werescored for the presence or absence of SARS-CoV-2 cytopathic effects(CPE) in the cells. The titer was the inverse of the last dilution ofdantrolene that inhibits the viral infection (cells that do not displayCPE), i.e., the lowest effective titer was the last dilution ofdantrolene that consistently inhibited viral infection (cells do notdisplay CPE) across all time points.

There were several controls present on each plate for the VN assay.First, there was a compound control that lacked virus to ensure that thecompound itself did not cause CPE; this control was performed using thelowest dilution of compound in the series (usually 1:10) and additionalserial dilutions at the test concentrations. There were also negativecontrol wells (without compound or virus) to verify that the serum-freemedia did not cause CPE. Also, a back-titer of the virus was performedwhich acted as a positive CPE control for the virus, and it served toverify that the titer of the standardized virus was within acceptablerange. Results from the samples on that plate were considered valid ifall of these controls met their acceptance criteria.

Without wishing to be bound to any particular theory, it is believedthat dantrolene modulates intracellular Ca, including by mechanisms notpreviously reported, thereby affecting the ability of the SARS-CoV-2virus to, for example, infect cells, replicate, mature, create virions,or release from cells.

Example 3

Dantrolene concentrations were tested: 5, 10, 20, 30, 40, 50 and 100 μM(as RYANODEX, dantrolene sodium).

Testing against a SARS-CoV-2 virus, represents the etiologic agent ofthe COVID-19 global pandemic. Testing was via a standard virusneutralization (VN) assay, which assessed ability to neutralizeSARS-CoV-2.

Virus Neutralization (VN) Assay. The VN assay was performed using VeroE6 cells, an African green monkey cell line, which are susceptible toSARS-CoV-2 infection. Vero E6 cells were cultured in growth media(Dulbecco's Modified Eagle Medium supplemented with 5% FBS (fetal bovineserum), Glutamax, and PSN (penicillin, streptomycin, and neomycin)). Thecells were seeded into deep 96-well plates one day prior to the VN assayand incubated at 37° C. and 5% CO₂ to allow the cells to grow to 70%confluency. Each of the samples and controls were performed intriplicate.

The preparation of RYANODEX for VN was performed the day of assay.RYANODEX was reconstituted with 5 mL of sterile water for injection, asdescribed in the RYANODEX Prescribing Information, to prepare an initialstock having a dantrolene concentration of 50 mg/mL. The stock wasfurther diluted to a dantrolene concentration of 100 μM using cellgrowth media and then serially diluted to 50, 40, 30, 20, 10 and 5 μM.The dilutions were preincubated on cells for 60 minutes prior toaddition of virus.

Standardization of the virus required that a Medium Tissue CultureInfectious Dose (TCID50) have been previously performed to determine theconcentration of infectious virus particle per mL of virus stock, usingprocedures known in the art. See, e.g., Reed & Muench, (1938) A simplemethod of estimating fifty percent endpoints, The American Journal ofHygiene. 27: 493-497; World Health Organization, Laboratory Procedures,Serological detection of avian influenza A(H7N9) infections bymicroneutralization assay, May 23, 2013 Using the TCID50 titer,calculations were performed to define how much serum-free media to addto the virus stock to yield 1e2 TCID50/mL. Once the standardized viruswas made, an equal volume of it was added to the deep 96-well platecontaining the diluted compound samples. Virus was added to theappropriate wells and incubated with cells and compound for 2 hours. Thecells were then washed 3 times with fresh media and 100 μL/well of freshmedia was added to all wells and further incubated for 6 days. Duringthe incubation period, the wells were observed under a phase contrastinverted scope and were scored for the presence or absence of SARS-CoV-2cytopathic effects (CPE) in the cells. The titer was the inverse of thelast dilution of dantrolene that inhibits the viral infection (cellsthat do not display CPE), i.e., the lowest effective titer was the lastdilution of dantrolene that consistently inhibited viral infection(cells do not display CPE) across all time points.

There were several controls present on each plate for the VN assay.First, there was a dantrolene control that included RYANODEX alone atthe test concentrations without virus to ensure that dantrolene itselfat the tested concentrations does not cause CPE. There were alsonegative control wells (without RYANODEX or virus) to verify that theserum-free media did not cause CPE. Also, a back-titer of the virus thatincluded 100 TCID50/well of virus was performed which acted as apositive CPE control for the virus, and it served to verify that thetiter of the standardized virus was within acceptable range. Resultsfrom the samples on that plate were considered valid if all of thesecontrols met their acceptance criteria.

The analysis of the neutralization assay revealed that the highestdantrolene concentrations (50 and 100 μM dilution) showed cytopathiceffects on Days 2 and 4, in both infected cells and uninfected controls,as expected. The 50 μM dilution had rebound cell growth on Day 6post-infection, but it was decreased compared to the lower dilutions.The uninfected controls displayed the same cytotoxicity profile as theinfected replicates at these higher dantrolene concentrations.

On Day 2 post-infection no CPE was observed in any wells containingRYANODEX. On Day 4 and Day 6 post-infection, no CPE was observed incells incubated with 20-40 μM of dantrolene. CPE was observed in 2 of 3replicates at Days 4 and 6 post-infection with 10 μM of dantrolene, butno CPE was observed at Day 2. At the 5 μM concentration of dantrolene,CPE was observed in 1 of 3 replicates at Days 4 and 6 post infection,but no CPE was observed at Day 2 at this dantrolene concentration. SeeTable.

TABLE Dilution Day 2 ReaD Day 4 Read Day 6 Read 100 μM 3/3 wells had >90% CPE 3/3 wells had > 90% CPE 3/3 wells had > 90% CPE  50 μM 3/3 wellshad sparse 3/3 wells had sparse 3/3 wells had sparse confluencyconfluency confluency but greater than Day 4  40 μM 3/3 wells had No CPE3/3 wells had No CPE 3/3 wells had No CPE  30 μM 3/3 wells had No CPE3/3 wells had No CPE 3/3 wells had No CPE  20 μM 3/3 wells had No CPE3/3 wells had No CPE 3.3 wells had No CPE  10 μM 3/3 wells had No CPE2/3 wells had > 90% CPE 2/3 wells had > 90% CPE  5 μM 3/3 wells had NoCPE 1/3 wells had > 90% CPE 1/3 wells had > 90% CPE CPE = rytopathiceffects

All uninfected controls remained healthy and did not display any CPEthroughout the 6-day post-infection incubation period. It was concludedthat the minimum inhibitory concentration of dantrolene is 20 μM, thoughlower concentrations of 10 and 5 μM also showed anti-viral activity. Nocytopathic effects (indicating no virus growth) were observed with the20 and 40 μM dantrolene concentrations at all timepoints. Cytotoxiceffects were only observed at the 2 highest dantrolene concentrations(100 and 50 μM), both with or without virus. In contrast, at the lowerdilutions (<50 μM dantrolene) uninfected Vero E6 cells showed good cellviability. The control cells infected with the virus but withoutRYANODEX all had CPE, evidencing viral growth.

In summary, the VN assay demonstrated the in vitro antiviral activityand lack of cytotoxicity of RYANODEX at dantrolene concentrationscompatible with human plasma levels observed after administration of therecommended doses of RYANODEX.

Without wishing to be bound to any particular theory, it is believedthat dantrolene modulates intracellular Ca²⁺, including by mechanismsnot previously reported, thereby affecting the ability of the SARS-CoV-2virus to, for example, infect cells, replicate, mature, create virions,or release from cells.

Example 4 Methodology

The study is a single-center, open-label, two-arm study of dantrolenefor the adjuvant treatment of SARS administered intravenously (IV). Inone treatment arm, dantrolene will be administered in conjunction withcurrent standard of care following medical practice and proceduresestablished for the treatment of patients with SARS. In the secondtreatment arm, subjects will receive current standard of care followingmedical practice and procedures established for the treatment ofpatients with SARS.

Following initial triage and primary assessment of a subject, thesubject's baseline status will be documented. Once eligibility criteriaand baseline status are obtained, administration of dantrolene will beinitiated.

Group A: dantrolene, in addition to standard of care

Group B: standard of care only

Treatment Administration—Group A

Eligible SARS subjects randomized to Group A will receive dantrolene (asRYANODEX, dantrolene sodium) as follows:

-   -   A 1 mg/kg dantrolene dose will be administered as IV push        approximately every 12 hours for 2 consecutive days (Study Day 1        and Day 2).    -   On Day 3 of the Study, subjects showing adequate tolerability to        1 mg/kg dose will start receiving 2 mg/kg as IV push        approximately every 12 hours for the remainder of the study        (Study Day 3 to Day 14, inclusive). Adequate tolerability is        defined as lack of clinically significant adverse reactions that        may have a negative impact on the subject's overall status that        are not secondary to SARS, any other underlying condition or        concomitant medication.    -   If a patient does not show adequate tolerability to the 2        mg/dose, the subject will be discontinued from the study.    -   Each subject participating in the study will receive up to        four (4) 1 mg/kg doses (Days 1-2) and up to 24 (twenty four)        doses of 2 mg/kg (Days 3-14), each administered as an IV push        (up to a minute).

Subjects receiving dantrolene will continue to receive all othertreatments as prescribed, with the exception of treatments included inthe Exclusion Criteria. Assessment of vital signs (blood pressure, heartrate, respiratory rate, body temperature) should be clearly recordedprior to and within 10 minutes after administration of each dose.

Group B

Patients randomized to Group B will receive standard of care, followingacceptable medical practice.

Study Phases

The study will include 2 phases: Screening and Treatment.

During the Screening Phase, eligibility and baseline assessment will beperformed. Informed Consent will be obtained prior to initiation ofstudy procedures.

After determination of eligibility and obtaining Informed Consent,eligible patients will be randomized to Group A or Group B and theTreatment Phase will be initiated and will proceed with administrationof Study Drug as indicated above to Group A. Non-eligible subjects willreceive medical assistance as deemed necessary by the attendingphysician following accepted medical practices.

Stopping the Study Drug

If the subject demonstrates clinically significant signs/symptoms ofdantrolene toxicity, the study drug should be stopped. Dantrolenetoxicity may include muscular weakness and alterations in the state ofconsciousness (e.g., lethargy, sedation), vomiting, diarrhea, andcrystalluria, which are not attributable to other cause, such asprogression of SARS, other underlying conditions (e.g., sepsis, hypoxia,uncontrolled diabetes) and/or concomitant medications (e.g., sedatives,antibiotics, antipyretics). The study drug can be stopped at any time.

Diagnosis and Main Criteria for Inclusion

Male or non-pregnant female subjects will be entered into the study ifthey are diagnosed with SARS and meet all the following criteria atScreening:

-   -   At least 18 years of age.    -   Willing and able to provide written informed consent prior to        performing study procedures, or an authorized representative is        willing and able to provide consent on behalf of the patient if        he/she is unable to do so.    -   SARS severity score 3-5 according to the WHO Ordinal Scale of        Severity    -   SARS symptoms onset within 7 days prior to Screening.    -   Has laboratory-confirmed SARS-CoV infection as determined by        PCR, or other commercial or public health assay within 48 hours        prior to Screening.    -   Febrile defined as temperature ≥36.6° C. armpit, ≥37.2° C. oral,        or ≥37.8° C. rectal documented at least within 48 hours of        consent.

Male or non-pregnant female subjects will be excluded from entering thisstudy if they meet any of the following criteria at Screening:

-   -   Participation in any other clinical trial of an experimental        treatment for SARS.    -   Alanine Aminotransferase (ALT) or aspartate aminotransferase        (AST) >5× upper limit of normal (ULN).    -   Pregnant women or women who are breastfeeding.    -   Presence of comorbidities that imply a poor prognosis (according        to clinical judgment).    -   Allergy to dantrolene.

Dosage and Mode of Administration

Ryanodex: (dantrolene sodium) for injectable suspension; 250 mg/vial tobe reconstituted with 5 mL of sterile water for injection (without abacteriostatic agent) to yield a 50 mg/mL suspension; to be administeredas a rapid IV push of 1 mg/kg, as described in the protocol.

Efficacy will be evaluated on criteria including:

-   -   World Health Organization (WHO) Ordinal Scale of Severity score.    -   Sequential Organ Failure Assessment (SOFA) score.    -   Length of time to normalization of fever (fever normalization as        defined by temperature <36.6° C. armpit, <37.2° C. oral, or        <37.8° C. rectal sustained for minimum of 24 hours).    -   Length of time to normalization of oxygen saturation (oxygen        normalization as defined by peripheral capillary oxygen        saturation (SpO2) >94% sustained for at least 24 hours).    -   Change in clinical status at Day 14 using the WHO Ordinal Scale        (1-8 score), compared to Baseline.        -   1: Ambulatory. No limitations of activities        -   2: Ambulatory. Limitation of activities        -   3: Hospitalized, Mild Disease. No oxygen therapy        -   4: Hospitalized, Mild Disease. Oxygen by mask or nasal            prongs        -   5: Hospitalized, Severe Disease. Non-invasive ventilation or            high-flow oxygen        -   6: Hospitalized, Severe Disease. Intubation and mechanical            ventilation        -   7: Hospitalized, Severe Disease. Ventilation+additional            organ support (pressors, renal replacement therapy,            extracorporeal membrane oxygenation)        -   8: Death    -   Change in clinical status at Day 5 and Day 10 using the WHO        Ordinal Scale (1-8 score), compared to Baseline.    -   Time to a 1-point decrease in the WHO Ordinal Scale score.    -   Change in Sequential Organ Failure Assessment (SOFA) daily score        (Days 1-14) compared to Baseline.    -   Length of time to normalization of fever (fever normalization as        defined by temperature <36.6° C. armpit, <37.2° C. oral, or        <37.8° C. rectal sustained for minimum of 24 hours).    -   Length of time to normalization of oxygen saturation (oxygen        normalization as defined by peripheral capillary oxygen        saturation (SpO2) >94% sustained for at least 24 hours).

Safety will be evaluated and will include measurement and observation of(if any):

-   -   Vital signs (heart rate, blood pressure, respiratory rate, body        temperature)    -   Clinical laboratory tests (hematology and blood chemistry)    -   ECG monitoring    -   Oxygen saturation    -   Physical exam

Example 5

Dantrolene concentrations are tested: 5, 10, 20, 30, 40, 50 and 100 μM(as RYANODEX, dantrolene sodium).

Testing is via a standard virus neutralization (VN) assay, whichassesses ability to neutralize SARS-CoV.

Virus Neutralization (VN) Assay. The VN assay is performed using cellswhich are susceptible to SARS-CoV infection. The cells are seeded into96-well plates one (1) to three (3) days prior to VN assay, and areincubated at 37° C. and 5% CO₂. On the day of assay, the monolayer ofcells is at least 70% to 80% confluent in order to run the VN. Thepreparation of the compound for VN is performed the day of assay. Thecompound is diluted with serum-free media to the desired startingconcentration (1:10) and then serially diluted 2-fold in serum-freemedia.

Standardization of the virus requires that a TCID50 has previously beenrun to determine the concentration of infectious virus particle per mLof virus stock. Using the TCID50 titer, calculations are performed todefine how much serum-free media to add to the virus stock to yield 1e2TCID50/mL. Once the standardized virus is made, an equal volume of it isadded to the deep 96-well plate containing the diluted compound samples.Incubation is maintained for at least one hour. The virus/compoundmixture is then transferred from the deep 96-well racks into theappropriate cell-seeded 96-well plates. After this addition, the platesare returned to the 37° C. and 5% CO₂ incubator for three (3) to five(5) days to six (6) days. After the incubation period, the wells areobserved under a phase contrast inverted scope and are scored for thepresence or absence of SARS-CoV cytopathic effects (CPE) in the cells.The titer is the inverse of the last dilution of serum that inhibits theviral infection (cells that do not display CPE), i.e., the lowesteffective titer was the last dilution of dantrolene that consistentlyinhibited viral infection (cells do not display CPE) across all timepoints.

There are several controls present on each plate for the VN assay.First, there is a compound control that lacks virus to ensure that thecompound itself does not cause CPE; this control is performed using thelowest dilution of compound in the series (usually 1:10) and additionalserial dilutions at the test concentrations. There are also negativecontrol wells (without compound or virus) to verify that the serum-freemedia does not cause CPE. Also, a back-titer of the virus is performedwhich acts as a positive CPE control for the virus, and it serves toverify that the titer of the standardized virus is within acceptablerange. Results from the samples on that plate are considered valid ifall of these controls meet their acceptance criteria.

Without wishing to be bound to any particular theory, it is believedthat dantrolene modulates intracellular Ca²⁺, including by mechanismsnot previously reported, thereby affecting the ability of SARS-CoV to,for example, infect cells, replicate, mature, create virions, or releasefrom cells.

Example 6

Dantrolene concentrations are tested: 5, 10, 20, 30, 40, 50 and 100 μM(as RYANODEX, dantrolene sodium).

Testing is via a standard virus neutralization (VN) assays known in theart that assess the ability to neutralize SARS-CoV. The VN assay isperformed using methods known in the art to measure the ability of avirus to infect cells, replicate, mature, create virions, and releaseactive virus from cells.

Typically, VN assays require a compatible host cell that is susceptibleto virus infection. Suitable cells lines include, but are not limitedto, Vero E6, HeLa, 293T, L929, fibroblasts, CHO, B95-8, MRC-5, HEp-2,SPF CE, MDCK, COS-7, and LLC-MK2 Derivative. In other example, primarycells isolated from an animal can be used in the assay. Cells used forVN assays can be wild-type cells, or they can be cells that have beengenetically modified using techniques known in the art. Cells lines arecultured in an appropriate growth media, including, but not limited toDulbecco's Modified Eagle Medium (DMEM), RPMI, Eagle's Minimum EssentialMedium (EMEM), Leibovitz's L-15 Medium, and VeroPlus SFM. Such growthmedia contains supplements required to sustain grown of the cells.Exemplary supplements include, but are not limited to fetal bovineserum, glutamax, penicillin, streptomycin, and neomycin. In otherexamples, supplements can be added to select for cells with specificgenetic modifications.

The cells are seeded into deep 96-well plates prior to the VN assay andincubated at 37° C. and 5% CO₂ to allow the cells to grow to anappropriate confluency. Each of the samples and controls are performedin triplicate.

The preparation of RYANODEX for VN is performed the day of assay.RYANODEX is reconstituted with 5 mL of sterile water for injection, asdescribed in the RYANODEX Prescribing Information, to prepare an initialstock having a dantrolene concentration of 50 mg/mL. The stock isfurther diluted to a dantrolene concentration of 100 μM using cellgrowth media and then serially diluted to 50, 40, 30, 20, 10 and 5 μM.The dilutions are preincubated on cells for 60 minutes prior to additionof virus.

Standardization of the virus requires that a Medium Tissue CultureInfectious Dose (TCID50) have been previously performed to determine theconcentration of infectious virus particle per mL of virus stock, usingprocedures known in the art. See, e.g., Reed & Muench, (1938) A simplemethod of estimating fifty percent endpoints, The American Journal ofHygiene. 27: 493-497; World Health Organization, Laboratory Procedures,Serological detection of avian influenza A (H7N9) infections bymicroneutralization assay, May 23, 2013. Using the TCID50 titer,calculations are performed to define how much serum-free media to add tothe virus stock to yield 1e2 TCID50/mL. Once the standardized virus ismade, an equal volume of it is added to the deep 96-well platecontaining the diluted compound samples. Virus is added to theappropriate wells and incubated with cells and compound for anappropriate amount of time consistent with observations from the TCID50assay. In some cases, this time period is 2 hours. In other cases, thetime period is 30 minutes to 1 hour. In other instances, the time periodis 4-8 hours. In yet other instances, the virus may be incubated withthe cells for 24-48 hours. And in other instances, cells may continue tobe incubated with virus over the duration of the assay. Further,infection may be facilitated by other methods known in the art, such asspin infection.

If the virus is removed from the cells prior to scoring for anti-viraleffects, the cells are then washed 3 times with fresh media and 100μL/well of fresh media is added to all wells and further incubated foran appropriate length of time depending on the VN assay being employed.

At an appropriate time, the cells are assayed for indications of viralinfection and replication using methods known in the art. By way ofexample only, the viral load of cells treated under different conditionsmay be measured using polymerase chain reaction (PCR) or other methodsknown in the art. By way of another example, changes to cellularmorphology, such as plaque formation, may be assayed. As yet anotherexample, wells containing virus are scored for the presence or absenceof viral cytopathic effects (VPE) in the cells using methods known inthe art, such as light microscopy or Annexin V, FITC, propidium iodide(PI), or haemotoxylin and eosin (H&E) staining. By way of yet anotherexample, chicken red blood cell suspensions are incubated with aserially diluted virus and monitoring for formation of a red blood celllattice in a hemagglutination assay. As another example, the attachmentof a suspension of red blood cells to the surface of cell monolayersinfected with virus is monitored in a hemadsorption assay.

The titer is the inverse of the last dilution of dantrolene thatinhibits the viral infection, i.e., the lowest effective titer is thelast dilution of dantrolene that consistently inhibits viral infectionacross all time points.

There are several controls present on each plate for the VN assay.First, there is a dantrolene control that includes RYANODEX alone at thetest concentrations without virus to ensure that dantrolene itself atthe tested concentrations does not cause a positive signal using theassay for viral infection. There are also negative control wells(without RYANODEX or virus) to verify that the serum-free media does notcause a positive signal using the assay for viral infection. Also, aback-titer of the virus that includes 100 TCID50/well of virus isperformed which acts as a positive VPE or viral infection control, andit serves to verify that the titer of the standardized virus is withinacceptable range. Results from the samples on that plate are consideredvalid if all of these controls meet their acceptance criteria.

Without wishing to be bound to any particular theory, it is believedthat dantrolene modulates intracellular Ca²⁺, including by mechanismsnot previously reported, thereby affecting the ability of SARS-CoV to,for example, infect cells, replicate, mature, create virions, or releasefrom cells.

1. A method of treating Severe Acute Respiratory Syndrome in a subjectcomprising administering to the subject dantrolene or a pharmaceuticallyacceptable salt thereof or by administering a dantrolene prodrug or apharmaceutically acceptable salt thereof.
 2. The method of claim 1,comprising administering dantrolene to the subject.
 3. The method ofclaim 1, comprising administering a pharmaceutically acceptable salt ofdantrolene to the subject.
 4. The method of claim 1, comprisingadministering dantrolene sodium to the subject.
 5. The method of claim1, wherein the administration results in at least a 1-point decrease inthe subject's WHO Ordinal Scale score, as compared to baseline.
 6. Themethod of claim 1, wherein the administration results in an improvementin the subject's Sequential Organ Failure Assessment daily score, ascompared to baseline.
 7. The method of claim 1, wherein theadministration results in a reduction of time to normalization of feverin the subject, as compared to the amount of time to normalization offever in a control subject.
 8. The method of claim 1, wherein theadministration results in a reduction of time to normalization of oxygensaturation in the subject, as compared to the amount of time tonormalization of oxygen saturation in a control subject
 9. A method forinhibiting replication of SARS-CoV in a subject comprising administeringto the subject dantrolene, or a pharmaceutically acceptable salt thereofor by administering a dantrolene prodrug or a pharmaceuticallyacceptable salt thereof.
 10. The method of claim 9, comprisingadministering dantrolene to the subject.
 11. The method of claim 9,comprising administering a pharmaceutically acceptable salt ofdantrolene to the subject.
 12. The method of claim 9, comprisingadministering dantrolene sodium to the subject.
 13. A method forinhibiting replication of SARS-CoV in a host cell, for inhibiting entryof SARS-CoV into a host cell, for inhibiting SARS-CoV virion maturationin a host cell, or for inhibiting release of SARS-CoV from a host cellcomprising administering to the host cell dantrolene, or apharmaceutically acceptable salt thereof or by administering adantrolene prodrug or a pharmaceutically acceptable salt thereof. 14.The method of claim 13, comprising administering dantrolene to the hostcell.
 15. The method of claim 13, comprising administering apharmaceutically acceptable salt of dantrolene to the host cell.
 16. Themethod of claim 13, comprising administering dantrolene sodium to thehost cell.
 17. A method for reducing the infectivity of SARS-CoV byadministering to a host cell dantrolene, or a pharmaceuticallyacceptable salt thereof or by administering a dantrolene prodrug or apharmaceutically acceptable salt thereof.
 18. The method of claim 17,comprising administering dantrolene to the host cell.
 19. The method ofclaim 17, comprising administering a pharmaceutically acceptable salt ofdantrolene to the host cell.
 20. The method of claim 17, comprisingadministering dantrolene sodium to the host cell.